Lipopolysaccharides are potential endotoxins, i.e. toxic, natural compounds found inside pathogens such as bacteria. Classically, an “endotoxin” is a toxin, which unlike an “exotoxin”, is not secreted in soluble form by live bacteria, but is a structural component in the bacteria which is released mainly when bacteria are lysed.
Lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) is a prototypical example of an endotoxin that is found in the outer membrane of various gram-negative bacteria. The term LPS is often used interchangeably with endotoxin, owing to its historical discovery. In the 1800s it became understood that bacteria could secrete toxins into their environment, which became broadly known as “exotoxin”. The term endotoxin came from the discovery that portions of gram-negative bacteria itself can cause toxicity, hence the name endotoxin. Studies of endotoxin over the next 50 years revealed that the effects of “endotoxin” was in fact due to lipopolysaccharide. The only gram-positive bacteria known to produce endotoxin is Listeria monocytogenes. 
LPS consist of a polysaccharide (sugar) chain and a lipid moiety, known as lipid A, which is responsible for the toxic effects. The polysaccharide chain is highly variable amongst different bacteria. LPSs are approximately 10 kDa in size but can form large aggregates up to 1000 kDa. Humans are able to produce antibodies to LPSs after exposure but these are generally directed at the polysaccharide chain and do not protect against a wide variety of endotoxins. Injection of a small amount of LPS in human volunteers produced fever, a lowering of the blood pressure, and activation of inflammation and coagulation. LPSs are in large part responsible for the dramatic clinical manifestations of infections with pathogenic gram-negative bacteria, such as Neisseria meningitidis, the pathogen that causes fulminant meningitis.
In pharmaceutical production, it is necessary to remove LPSs from drug product containers as even small amounts of this endotoxin will cause illness, but not disease, in humans. Usually, a depyrogenation oven is used for this purpose. Temperatures of approximately 400° C. are required to break down this substance. Based on primary packaging material as syringes or vials a glass temperature of 250° C. and a holding time of 30 min is typical to achieve 3 log reduction on endotoxin levels.
Likewise, it must be ensured that endotoxins are removed from pharmaceutical grade biopolymers, such as alginate, xanthan gum and gelatine. To this end several methods have been proposed in the prior art.
WO 93/13136 describes a process for purifying polysaccharides comprising:                filtering a polysaccharide solution having a concentration of less than 1% through a first nitrocellulose filter having a pore size of at least 45 microns;        filtering the resulting solution through a second nitrocellulose filter having a pore size of less than 12 microns;        filtering the resultant solution through a membrane having a pore size less than 12 microns, said membrane modified with polypeptides to bind hydrophobic impurities; and        dialyzing the resulting solution with a membrane having a lower molecular weight cut-off than the first ultra-filtration membrane.        
The process described in WO 93/13136 is not suitable for processing viscous solutions and is not very robust.
U.S. Pat. No. 5,589,591 describes a method of making a highly purified, substantially endotoxin-free arabinogalactan composition which comprises the steps of:                (i) removing from an arabinogalactan-containing preparation by ultrafiltration, materials of a molecular weight that are less than the molecular weight of the arabinogalactan composition, and collecting the arabinogalactan-containing fraction thereof;        (ii) thereafter removing from the arabinogalactan-containing fraction by ultrafiltration, endotoxin and materials of a molecular weight that are greater than the molecular weight of the arabinogalactan; and        (iii) collecting the resulting arabinogalactan-containing fraction, which has been rendered substantially endotoxin-free.        
What has been said above in relation to WO 93/13136 equally applies to this US patent.
WO 00/09566 describes a method for obtaining ultra-pure alginate, said method comprising the steps of:                extracting a dissolved material consisting of algae or raw alginate using a complexing agent such EDTA or activated carbon;        filtering the solution;        precipitating the alginate contained in the solution; and        recovering the precipitated alginate.WO 00/09566 also describes an embodiment in which prior to filtration cellular components and particles are sedimented with the aid of porous binding agents such as kieselguhr, cellulose or recycling materials from renewable sources. The process described in the international patent application suffers from the drawback that the porous binding agents are difficult to precipitate from viscous fluids, even at increased centrifugal force. If such viscous fluids additionally exhibit a high specific density, it is nearly impossible to separate the insoluble material from the aquous phase.        
U.S. Pat. No. 6,451,772 discloses a method for preparing a biopolymer composition comprising a salt of a biopolymer having an endotoxin content less than about 100 endotoxin units per gram comprises the steps of                (i) contacting an aqueous solution of a biopolymer salt with a hydrophobic material, such as polystyrene, polypropylene or fluorocarbon polymers, to adsorb endotoxin on said material; and        (ii) precipitating the biopolymer salt having an endotoxin content less than about 100 endotoxin units per gram from the solution by mixing a water miscible organic solvent with the solution.The US patent also discloses a method in which the precipitation step is replaced by a liquid-liquid extraction using a water immiscible solvent. Furthermore, it is mentioned that in case of alginates the aqueous biopolymer solution may be contacted with activated carbon to remove polyphenols. Important drawbacks of the method described in U.S. Pat. No. 6,451,772 are that (i) it is virtually impossible to avoid co-precipitation of the biopolymer salt and the hydrophobic material upon addition of the water miscible organic fluid and (ii) poor mass transfer during liquid-liquid extraction.        
EP-A 0 072 513 describes a process for the preparation of an antigenic capsular bacterial polysaccharide from a culture medium or a solution comprising at least one precipitation step by formation of a complex between a polyionic derivative of the medium—which polyionic derivative is at least said polysaccharide—and a quaternary ammonium salt, wherein at least one precipitation of the complex is performed on an inert porous support constituted of cellulose, an alkaline-earth metal salt substantially insoluble in water, aluminium oxide or hydroxide, silicon oxide, a silicate or an aluminosilicate. In Example 1 of the European patent application Celite® 545 (kieselguhr) and Cetavlon® (a cationic surfactant) are added to a fermentate containing bacterial polysaccharide. After 5 hours, a complex of polysaccharide/Cetavlon® and Celite® 545 is isolated by decanting the supernatant.
O. Adam et al. (Anal. Biochem 225 (1995), 321-327 describes a method for removing endotoxins from exopolysaccharides by temperature-induced Triton X-114 phase separation. D. Petsch & F. B. Anspach, Journal of Biotechnology 76 (2000), 97-119 describes a method for Triton X-114 based removal of endotoxins as well. The processes described in these papers have several disadvantages. Due to the heterogenic nature of the endotoxin and the equilibrium distribution of LPS in the liquid 2-phase systems, the final endotoxicity reduction of the alginate is limited. Furthermore, the method is not suitable for processing viscous fluids unless very high centrifugal forces are applied to separate the phases. Finally, it is very difficult to quantitatively separate the upper liquid phase from the lower liquid phase, making the process not very robust.